title: Rad GTPase inhibits cardiac fibrosis through connective tissue growth factor.
abstract: OBJECTIVE
  Our previous studies documented that Rad (Ras associated with diabetes), a member of the RGK (Rad, Gem, and Kir) family of Ras-related small G protein, is significantly decreased in human failing hearts and plays an important role in attenuating cardiac hypertrophy. The goal of this study is to identify the effect of Rad on cardiac fibrosis and the underlying mechanisms.
  
  
  RESULTS
  Rad knockout (KO) mice showed more severe cardiac fibrosis compared with wild-type littermate controls as detected by Sirius Red staining. Western blot analyses demonstrated that the expression of connective tissue growth factor (CTGF), a key mediator of fibrosis, increased dramatically in Rad KO mice. Overexpression of Rad in cultured neonatal cardiomyocytes suppressed both basal and transforming growth factor-β1-induced CTGF expression. Elevated CTGF expression was observed in cardiomyocytes when Rad was reduced by RNA interference. Moreover, cardiac fibroblasts produced greater extracellular matrix (ECM) when stimulated with conditioned medium from Rad-knockdown cardiomyocytes. ECM production was completely abolished by adding a CTGF-neutralizing antibody into the medium. CCAAT/enhancer-binding protein δ (C/EBP-δ) was demonstrated to activate CTGF in cardiomyocytes. Chromatin immunoprecipitation assay and co-immunoprecipitation further demonstrated that Rad inhibited the binding of C/EBP-δ to the CTGF promoter via direct interaction with C/EBP-δ.
  
  
  CONCLUSIONS
  Our data reveal that Rad deficiency can lead to cardiac fibrosis. Rad inhibits CTGF expression through binding with C/EBP-δ, thus regulating ECM production in the heart. This study suggests a potential link between decreased Rad levels and increased cardiac fibrosis in human failing hearts.
authors.affiliation: Institute of Molecular Medicine, Peking University, No. 5, Yi He Yuan Road, Beijing 100871, China.
authors.forename: Ji
authors.initials: J
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Chang
authors.forename: Chunlei
authors.initials: C
authors.lastname: Chen
authors.forename: Meiling
authors.initials: M
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Luo
authors.forename: Milton
authors.initials: M
authors.lastname: Hamblin
authors.forename: Luis
authors.initials: L
authors.lastname: Villacorta
authors.forename: Jing-Wei
authors.initials: JW
authors.lastname: Xiong
authors.forename: Y Eugene
authors.initials: YE
authors.lastname: Chen
authors.forename: Jifeng
authors.initials: J
authors.lastname: Zhang
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000378:metabolism; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000378:metabolism*; D005355:Fibrosis; D006333:Heart Failure / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000473:pathology; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D012720:Severity of Illness Index; D015398:Signal Transduction; D014162:Transfection; D018631:ras Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; C405462:CCAAT-enhancer-binding protein-gamma; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; C526316:CCN2 protein, rat; D017077:Culture Media, Conditioned; C490030:Rrad protein, mouse; C490031:Rrad protein, rat; D055513:Connective Tissue Growth Factor; D018631:ras Proteins